PROJECT SUMMARY/ABSTRACT High iron stores are a well-defined risk factor for the pathogenesis of several diseases, including heart failure, liver cirrhosis, arthritis, diabetes and hypertriglyceridemia. Iron overload is best represented by hereditary hemochromatosis (HH), one of the most common genetic diseases in the North American Caucasian population, which is characterized by elevated intestinal absorption and progressive tissue deposition of iron. Polymorphisms in the HFE (High Fe) gene are the leading cause of HH, accounting for 7-32% in North American populations. Iron overload also occurs in blood transfusion, which is required for several anemias (e.g. thalassemia, sickle cell anemia) due to defects in blood cell metabolism. Notably, high iron stores in the brain are associated with several neurodegenerative diseases (e.g. Alzheimer?s and Parkinson?s diseases) and some pathological conditions, including traumatic brain injury. Iron chelators, such as deferoxamine and deferasirox, are clinically used to reduce iron burden, but the use of chelators is limited by a number of significant side effects, including agranulocytosis, neutropenia, ocular/auditory toxicities, musculoskeletal-joint pains, gastrointestinal disturbances and even death. Considering hundreds of millions of people affected by various types of iron overload, there are unprecedented needs for a new therapeutic strategy by controlling the transport of iron in the body. While the Divalent Metal Transporter 1 (DMT1) plays a well-established role in the absorption of iron as an essential nutrient from diet, up-regulation of intestinal DMT1 is associated with HH in both humans and mice. Since DMT1 is also required for red cell production in the bone marrow, a ?selective? suppression of intestinal DMT1 can be an excellent therapeutic target by direct delivery of DMT1 inhibitors to the site of absorption (i.e. oral administration) with no systemic effects. Although a few small molecule-based DMT1 inhibitors have been studied, overall enthusiasm is low because these inhibitors ?indirectly? alter DMT1 function, for example, by modifying redox status, as well as their unfavorable in vivo pharmacokinetic properties (poor solubility and rapid metabolism). Gene silencing has increased therapeutic potential to selectively decrease the levels of unwanted molecules, such as oncogenic proteins and pro-inflammatory cytokines. We have recently demonstrated that intestinal TNF? was significantly down-regulated in a mouse model of colitis after oral administration of siRNA in nanoparticles-in-microspheres (NMs), which improved colitis conditions. Thus, the major underlying hypothesis is that oral DMT1 silencing by siRNA-encapsulated NMs decreases intestinal uptake of dietary iron and improves iron overload and iron-mediated toxicity. The specific aims of this study are focused on 1) developing and validating DMT1 siRNA/NMs to inhibit intestinal iron transporters and iron absorption and 2) evaluating the therapeutic efficacy of DMT1 siRNA/NMs using a mouse model of iron overload. Overall, this strategy provides a selective and effective method to support therapeutic benefits over numerous iron overload disorders by a combination of siRNA and nanotechnology.